Activated Transcription Factor 3 in Association with Histone Deacetylase 6 Negatively Regulates MicroRNA 199a2 Transcription by Chromatin Remodeling and Reduces Endothelin-1 Expression

Abstract

P lacenta growth factor (PlGF), an angiogenic growth factor of the vascular endothelial growth factor (VEGF) family, has pleiotropic and redundant roles in development but features prominently in various pathological conditions such as ischemia, angiogenesis, inflammation, atherosclerosis, preeclampsia, and diabetic wound healing (1-9). PlGF expression is induced by hypoxia, erythropoietin, and iron; PlGF effects are manifested via binding to its sole cognate receptor, VEGF receptor 1 (VEGFR-1) (10-13).Recent studies have shown that plasma levels of PlGF are abnormally high in patients with hemolytic anemias, such as in sickle cell disease (SCD) (12,14). Moreover, high plasma PlGF levels correlate with increased incidence of vaso-occlusive events in SCD subjects (12). Consistent with these findings, plasma levels of plasminogen activator inhibitor 1 (PAI-1) and endothelin-1 (ET-1) are high in a mouse model of sickle cell disease, namely, Berkeley sickle (BK-SS) mice, which also show high plasma PlGF levels (15, 16). Conversely, PlGF knockout mice exhibit low plasma PlGF levels and low plasma levels of PAI-1 and ET-1 (15, 16). Furthermore, augmentation of erythroid PlGF expression in normal mice to the levels seen in sickle mice results in increased production of endothelin-1 with associated pulmonary changes, as seen in pulmonary hypertension (PH) in SCD (16); these results were corroborated in a study of 123 patients with SCD (16). Thus, studies in vitro and in vivo support the crucial role of PlGF in upregulating the expression of ET-1 in endothelial cells and associated pulmonary changes characteristic of pulmonary hypertension in SCD.We show that PlGF activates endothelial cells to upregulate the expression of genes such as the ET-1 and PAI-1 genes via activation of hypoxia-inducible factor 1 (HIF-1␣), independently of hypoxia (15,17). Moreover, we have shown that posttranscriptional regulation of ET-1 and PAI-1 is achieved by microRNA 199a2 (miR-199a2), which targets the 3= untranslated region (UTR) of HIF-1␣ mRNA (18).The DNM3 opposite strand (DNM3os) gene produces a noncoding RNA (ncRNA) that serves as the precursor of miR-199a2 and miR-214 (18). This locus is located within an intron of DNM3 and is transcribed from the opposite strand, hence, its designation. Transcription of DNM3os and of cotranscribed miR-199a2/miR-214 is greatly attenuated by PlGF, thus allowing unhindered expansion of HIF-1␣ activity and expression of genes, i.e., the ET-1 and PAI-1 genes, requiring this transcription factor, (18). However, the molecular mechanism of PlGF-mediated downregulation of miR-199a2/miR-214 and DNM3os ncRNA is not fully understood.In the present study, we show that repression of DNM3os in response to PlGF required the participation of activated transcription factor 3 (ATF3), which binds to ATF3 response elements in the DNM3os promoter. The ATF3 gene, a stress-inducible gene, has been shown to play important roles in several pathological conditions, including host immunity and cancer (19-21). To de- Citation...

Placenta growth factor mediated gene regulation in sickle cell disease

“…ATF3 has been shown to play an important role in the pathophysiology of diseases, including cancer, immunity and sepsis (80–82). Studies showed ATF3 binds to DNM3os promoter proximal cis-binding elements, located at −42/−35 nt and −816/−809 nt), as demonstrated by gain and loss of function of ATF3, DNM3os promoter-luciferase reporter assay and ChiP analysis (Figure 5)(79). Other studies have shown ATF3 can act either as a repressor or activator of transcription by recruitment of accessory proteins to the promoter element of genes (83).…”

“…PlGF treatment of cultured endothelial cells increased expression of activating transcription factor-3 (ATF3), a stress inducible gene (79). ATF3 has been shown to play an important role in the pathophysiology of diseases, including cancer, immunity and sepsis (80–82).…”

“…Following PlGF induction, proteomic analysis of endothelial cell lysates immunoprecipitated with antibody to ATF3 identified ATF3-interacting proteins (JDP2, ATF2 and HDACs). Since histone deacetylases (HDACs) are known to participate in gene repression by chromatin condensation following histone deacetylation (84), studies examined possible roles of HDACs 1,3,4,6,7,8,9 and 11 in PlGF-mediated repression of DNM3os utilizing commercially available HDAC shRNAs (79). Both HDAC6 and HDAC7 bound to the promoter of DNM3os along with JDP2 to cause repression of DNM3os transcription including pre-miR-199a2 (Figure 5).…”

“…Both Tubacin and TSA reversed PlGF-mediated repression of DNM3os transcription. The effect of Tubacin on derepression of DNM3os was locus specific as no effect was observed in transcription of DNM3 (sense strand) (79). Since histone marks, H3K27Ac and H3K9Ac are associated with chromatin remodeling at promoter and enhancer sites (85), studies examined the presence of such marks in the promoter of DNM3os proximal to the identified ATF3 sites.…”

“…Conversely, Tubacin antagonizes this change to maintain an open chromatin state at the promoter region of DNM3os. Moreover, PlGF induced secretion of ET-1 is antagonized by Tubacin treatment and HDAC6 shRNA, indicating that chromatin changes are associated with transcription of DNM3os/miR-199a2(Figure 5)(79). …”